(i) The effect of membrane composition on ethanol partitioning into lipid bilayers was assessed by headspace gas chromatography. A series of model membranes with different compositions have been investigated. Membranes were exposed to a physiological ethanol concentration of 20 mM. The concentration of membranes was 20 wt% which roughly corresponds to values found in tissue. Partitioning depended on the chemical nature of polar groups at the lipid-water interface. Compared to phosphatidylcholine, lipids with headgroups containing phosphatidylglycerol, phosphatidylserine, and sphingomyelin showed enhanced partitioning while headgroups containing phosphatidylethanolamine resulted in a lower partition coefficient. The molar partition coefficient was independent of a membranes hydrophobic volume. This observation is in agreement with our previously published NMR results which showed that ethanol resides almost exclusively within the membrane-water interface. At an ethanol concentration of 20 mM in water, ethanol concentrations at the lipid/water interface are in the range from 30 - 15 mM, corresponding to one ethanol molecule per 100-250 lipids. We obtained evidence for critical behavior in cholesterol-rich model membranes that form coexisting liquid ordered and disordered phases which have been linked to raft formation in biological membranes. Deuterium NMR was used to evaluate phase boundaries in cholesterol containing ternary lipid membranes. The precise thermodynamic description of phase behavior permitted to predict composition and temperature at which critical behavior occurs. NMR resonances are dramatically broadened in the vicinity of critical points confirming their existence. Broaden-ing was attributed to increased spin-spin relaxation rates arising from modulations of chain order on a microsecond timescale. We speculate that spectral broadening is a reflection of formation of lipid-cholesterol clusters with microsecond lifetimes. Critical fluctuations provide a mechanism to produce lipidic structures with submicron dimensions at physiologically relevant composition and temperatures. Work on this project has been a collaborative research effort between Dr. Sarah Veatch, Dr. Sarah Keller, and the NMR Section of LMBB. In the framework of this project we developed NMR tools for detection of ordered lipid domains in biological membranes that do not require isotopic labeling. In collaboration with Dr. Joshua Zimmerbergs laboratory at NIH, those tools have been used to search for ordered lipid domains in intact influenza virus. Evidence for coexistence of ordered and disordered lipid domains in both the intact virus envelope and in bilayers made from extracted viral lipid was obtained. The functional implications of formation of ordered lipid domains in viral membranes are under investigation. (ii) We considered the issue of whether or not shifts in the metarhodopsin I (MI) metarhodopsin II (MII) equilibrium from lipid composition are fully explicable by differences in bilayer curvature elastic stress. A series of six lipids with known spontaneous radii of monolayer curvature and bending elastic moduli were added at increasing concentrations to the matrix lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and the MI-MII equilibrium measured by flash photolysis followed by recording UV-vis spectra. The average area per lipid molecule and the membrane hydrophobic thickness were derived from measurements of the 2H NMR order parameter profile of the palmitic acid chain in POPC. For the series of ethanolamines with different levels of headgroup methylation, shifts in the MI-MII equilibrium correlated with changes in membrane elastic properties as expressed by the product of spontaneous radius of monolayer curvature, bending elastic modulus, and lateral area per molecule. However, for the entire series of lipids, elastic energy explained the shifts only partially. Additional contributions correlated with the capability of the ethanolamine headgroups to engage in hydrogen bonding with the protein, independent of the state of ethanolamine methylation, with introduction of polyunsaturated sn-2 hydrocarbon chains, and with replacement of the palmitic acid sn-1 chains by oleic acid. The experiments point to the importance of interactions of rhodopsin with particular lipid species in the first layer of lipids surrounding the protein as well as to membrane elastic stress in the lipid-protein domain. We explored if the surface of the GPCR rhodopsin should be viewed as homogeneous and the surrounding membrane as a continuum, or if specific interactions, in particular with polyunsaturated lipids may play a role in rhodopsin activation. The association of rhodopsin with poly- and monounsaturated lipids was studied by 1H MAS NMR with magnetization transfer from rhodopsin to lipid. It was shown that poly- and monounsaturated lipids interact specifically with different sites on rhodopsin. Associated lipids are in fast exchange with lipids of the matrix on a time-scale of milliseconds or shorter. All rhodopsin photointermediates transferred magnetization preferentially to DHA-containing lipids suggesting stronger interactions. Highest rates were observed for Meta-III rhodopsin. Interactions with DHA-containing lipids are headgroup dependent and strength increased in the sequence phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine. We used 1H MAS NMR on rhodopsin in intact rod outer segment disks to track changes in rhodopsin hydration upon photoactivation. The data indicate that metarhodopsin-I differs from dark adapted rhodopsin and other photointermediates by a higher level of hydration. Although these experiments do not directly determine where on the protein hydration changes, magnetization transfer is most efficient when the interactions are both short-range (<5 ) and long-lived (1-10 ns), conditions best satisfied by hydration of the protein core. Changes of GPCR hydration upon activation make GPCR signaling particularly sensitive to environmental factors that influence activity of water molecules. The same technique was applied to study hydration of membrane-embedded voltage-sensing protein domains in collaboration with the laboratory of Kenton Swartz, NINDS. (iii)Cannabinoid receptors have attracted much attention because of their role in health and disease including alcoholism. The peripheral cannabinoid receptor, CB2, is involved in immune and hormonal response. CB2 was expressed in Escherichia coli as a fusion with maltose-binding protein and several affinity tags. The fusion was cleaved and the receptor purified by Ni-NTA and Streptactin affinity chromatography in the presence of detergents. Several methods for reconstitution of CB2 into lipid bilayers (rapid dilution, dialysis, removal of detergents with absorbing resins) were compared. Composition, size, and homogeneity of proteoliposomes were investigated by analytical NMR, fluorescence spectroscopy using labeled lipid and CB2, dynamic light scattering, and sucrose gradient centrifugation. The protein was successfully stabilized during purification and reconstitution by a proper mixture of detergents, lipids, as well as ligand. Preparation of samples that contain milligrams of 90% functional CB2 was successful. The structural integrity of the reconstituted protein was confirmed by its ability to activate G-protein in response to agonist binding. We developed a bacterial fermentation protocol for production of a stable isotope-labeled cannabinoid receptor CB2 for subsequent structural studies of this protein by nuclear magnetic resonance spectroscopy. The fermentation process produced about 2 mg of purified, labeled and functional CB2 per liter of culture medium.